JP2002209462A - Production of protein in transgenic plant seed - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a transgenic plant producing seeds useful as a raw material for a feed abundantly containing phosphorus or carbohydrates. SOLUTION: This transgenic plant comprises a genomic DNA containing a promoter and a gene containing a nucleotide sequence encoding a hydrolase and operably connected to the promoter. The promoter drives the expression of the hydrolase in seeds during development of the transgenic plant, seeds during germination, germinated seeds or plant seed tissues of young plants grown from the germinated seeds.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to transgenic plants, and more particularly to transgenic plants that produce seeds useful as feedstocks for phosphorus or carbohydrate rich feeds.

[0002]

BACKGROUND OF THE INVENTION The use of transgenic plants for the production of pharmaceutical proteins and industrial enzymes has been proposed. Generally, expression of a recombinant protein relies on stable integration of the heterologous gene into the host plant genome, for example, using Agrobacterium-mediated transformation or particle bombardment. In terms of cost, the production of commercially valuable proteins using farm plants requires other biological production systems (eg, yeast cultures, bacterial cultures) that require complex and sophisticated maintenance bioreactors. Or mammalian cell culture). Further, protein production in plants can be easily scaled up to produce larger quantities.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a transgenic plant that produces seeds useful as a feedstock for phosphorus or carbohydrate rich feeds.

[0004]

SUMMARY OF THE INVENTION The present invention is a transgenic plant, wherein the genomic DNA comprises a promoter and a gene having a nucleotide sequence encoding an enzyme and operably linked to the promoter, Here, the present invention relates to a transgenic plant, wherein the promoter drives the expression of the hydrolase in a developing seed, a germinating seed, a germinated seed, or a seedling of a seedling of the transgenic plant.

More specifically, the present invention is as follows.

[0006] The present invention is a transgenic plant, wherein the genomic DNA comprises a promoter and a gene encoding a hydrolase and comprising a nucleotide sequence operably linked to the promoter. A transgenic plant is provided, wherein the promoter drives the expression of the hydrolase in a developing seed, a germinating seed, a germinated seed, or a seedling plant seed tissue of the transgenic plant.

In one embodiment, the hydrolase hydrolyzes a substrate naturally present in the plant seed tissue of the transgenic plant.

In another embodiment, the plant is a rice, barley, rye, oat, or rape plant.

[0009] In another embodiment, the promoter is
α-amylase gene promoter. Preferably, this promoter is the αAmy8 promoter. In yet another embodiment, the promoter is a glutelin gene promoter.

[0010] In another embodiment, the enzyme is a phytase. In yet another embodiment, the enzyme is amylopullulanase or α-amylase.

The present invention also provides a transgenic seed produced from the above-mentioned transgenic plant of the present invention.

[0012] In one embodiment, the seed is germinating.

[0013] The present invention also provides a method for producing a hydrolase, comprising the steps of providing the transgenic plant, collecting the transgenic seed from the transgenic plant, and providing the transgenic seed. Germinating to produce the hydrolase.

In one embodiment, the method for producing a hydrolase further comprises the step of isolating the hydrolase from the transgenic seed.

The present invention further provides a method for producing a polypeptide, the method comprising the steps of: genomic DNA of a transgenic plant encodes an α-amylase promoter, and the polypeptide and is operable by said promoter. Providing the transgenic plant comprising a gene comprising a nucleotide sequence operably linked;
Collecting a transgenic seed from the transgenic plant; germinating the transgenic seed to produce a mature seed; and isolating the polypeptide from the mature seed.

In one embodiment, in the method for producing a polypeptide described above, in the step of isolating, the polypeptide is isolated from an embryo or endosperm of the mature seed.

The present invention also provides a method for producing a polypeptide, comprising the steps of: genomic DNA of a transgenic plant encodes a glutelin gene promoter and a polypeptide and is operable by said promoter. Providing the transgenic plant comprising a gene comprising a nucleotide sequence linked to:
Collecting a transgenic seed from the transgenic plant; germinating the transgenic seed; and isolating the polypeptide from embryonic tissue or endosperm tissue of the seed.

The present invention also provides a method for preparing a transgenic plant, comprising the steps of: providing a plant with a promoter and a gene encoding a hydrolase and comprising a nucleotide sequence operably linked to the promoter; Wherein the promoter drives the expression of the hydrolase in a developing seed, a germinating seed, a germinated seed, or a seedling plant tissue of the transgenic plant. The process.

The present invention also provides a method for preparing a transgenic seed produced from the transgenic plant prepared by the above-described method for preparing a transgenic plant.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to the use of a transgene in a transgenic seed, wherein the hydrolase has a promoter active in plant seed tissue and drives the transcription of a sequence encoding this hydrolase. When expressed, it is based on the finding that it has a higher specific activity than expected (e.g., as compared to the E. coli producing enzyme).

More specifically, the invention features a transgenic plant (eg, a transgenic rice, transgenic barley, transgenic rye, or transgenic rape plant), wherein the genomic DNA of the transgenic plant is Promoter (for example, α-amylase promoter (for example,
αAmy8 gene promoter or glutelin promoter) and hydrolase, and include genes having nucleotide sequences operably linked to this promoter. This promoter drives the expression of hydrolase in developing seeds, germinating seeds, germinated seeds, or seedlings of transgenic plant seedlings. This enzyme hydrolyzes naturally occurring substrates in plant seed tissue as needed. If the enzyme is a phytase, the enzyme may also be an acid phosphatase (eg, an enzyme encoded by the appA gene), or Selenomonas rumi.
phytase (eg, an enzyme encoded by the phyA gene) from N. nanium (eg, strain JY35 available as ATCC 55785). This enzyme (for example, Thermoanaero
If it is amylopullulanase (from B. ethanolicus), the enzyme may also be an α-amylase. The present invention also includes transgenic seeds produced from the above transgenic plants. As described herein, a “seed” can be a developing seed, a mature seed, a germinating seed, a germinated seed, or a seedling. Accordingly, the present invention further provides a transgenic plant of the present invention, harvesting a transgenic seed from the transgenic plant, germinating the seed, and optionally, hydrolyzing the hydrolase from the transgenic seed. A method for producing a hydrolase by isolation is characterized.

The present invention also provides a transgenic plant, wherein the genomic DNA of the transgenic plant includes an α-amylase promoter and a gene encoding a polypeptide and having a nucleotide sequence operably linked to the promoter; Harvesting the transgenic seed from the transgenic plant; germinating the transgenic seed to produce a mature seed; and isolating the polypeptide from the mature seed (eg, from the embryonic tissue or endosperm tissue of the mature seed). And producing a polypeptide. Providing a transgenic plant, wherein the genomic DNA of the transgenic plant encodes a glutelin gene promoter and a polypeptide, and comprises a gene having a nucleotide sequence operably linked to the promoter; Methods for producing a polypeptide by harvesting; germinating the transgenic seed; and isolating the polypeptide from embryonic or endosperm tissue of the seed are also encompassed by the present invention.

As used herein, "phytase" is an enzyme that reacts with phytate or phytin to release phosphorus or a phosphorus-containing moiety that is absorbable in the GI of monogastric animals. is there. Therefore,
Inorganic phosphorus or phosphoric acid (phosphh) derived from phytic acid
The phosphatase that releases ate) is also a phytase.

One genetic element “operably linked” to the other element is one genetic element (either in a plus-strand, minus-strand, or double-stranded form) that connects the other genetic element. Means structurally configured to operate or affect it. For example, a promoter operably linked to a sequence encoding a polypeptide means that the promoter initiates transcription of a nucleic acid encoding the polypeptide.

Germination begins with the uptake of water by the seeds (inhibition) and the hypocotyl (usually radicle)
And ends with the start of elongation. Therefore, germination is
It does not encompass seedling growth, and seedling growth begins when germination has ended. For germination to be completed, the radicle must elongate and penetrate surrounding structures. A developing seed is a seed that is germinating or that supports seedling growth. As used herein, the term “seedling” refers to de Vogel “The Seedli”
ng ", Seedslings of Dicotile
dons, Center for Agricultu
ral Publishing and Documen
, Wageningen, Netherlands
ands, pages 9-25, 1983, means young plants grown from germinating seeds.

The α-amylase gene promoter and signal peptide sequence can be fused upstream of an enzyme open reading frame (ORF) or gene, which can be derived from a variety of sources. The chimeric gene is then introduced into the plant (eg, cereal) genome. For example, malt production that produces high levels of phytase (malte
d) The transgenic grains may be used for human consumption, animal feed, or food processing. Malt production is a process in which kernels are germinated under controlled conditions and in a contained facility to produce a consistent product.

The advantages of using transgenic malt-producing seeds to produce hydrolase as a diet include: (1) the seeds are easily transportable and resistant to breakage and are readily available; Contains large amounts of nutrients and minerals in form; (2) the nutrients in the seed are not chelated by, for example, phytic acid;
(3) In malted seeds, since malted seeds have long been used in the beer brewing and food industries and are known to be harmless to humans and animals, the Little toxicity is expected; and (4) the cost of producing monogastric animal feed is reduced because exogenous enzymes do not need to be added to the feedstock.

In addition, the transgenic plants of the present invention, or their organs and seeds, can be used to produce hydrolases for various industrial processes. Examples of such applications are in feed additives for non-ruminants, in soy processing, in food processing, or in the production of inositol or inositol phosphate from phytic acid.

[0029] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a transgenic plant that produces seeds useful as a feed for phosphorus or carbohydrate rich feed. The excellent properties of this seed and the resulting feed are due to the insertion of a transgene containing a promoter that is active in the seed tissue of the plant (and that highly expresses the enzyme in the seed tissue of the transgenic plant). Naturally, this enzyme
It can be expressed in either transgenic seeds or plants without affecting the advantages provided by the present invention. Seeds produced from such plants may have reduced phytic acid and increased dietary phosphorus content or reduced complex carbohydrates, as compared to a control plant without the transgene, and Simple carbohydrates can be increased.

Phytic acid (myo-inositol hexakisphosphate) is the major storage form of phosphorus in many seeds. Phytin is an insoluble mixture of potassium, magnesium and calcium salts of myo-inositol hexaphosphate (phytic acid). grain,
In fatty seeds and beans, phytate accumulates in the seeds during maturation and is 50% to 80% of the total phosphorus content of the seeds.
Account for%. Soy and corn flour are major components of animal feed. They include sufficient levels of phosphorus to meet the growth requirements of the animal, as long as the phytate-derived phosphorus is digestible (ie, absorbable) by the animal. Ruminants (multi-stomach animals) readily cleave phytic acid and release digestible phosphorus. However, monogastric animals (eg, pigs, poultry and fish) make very little use of phytic acid. Because they lack gastrointestinal (GI) tract enzymes that can hydrolyze phytate. This deletion requires supplementation of animal feed with phosphorus to meet dietary requirements. In addition, phytic acid chelates important cations (including iron, magnesium, manganese, zinc, calcium, copper and molybdenum) and phytic acid-cations-
Form protein complexes, thereby reducing the bioavailability of minerals and amino acids in feed. Thus, the present invention provides a means for releasing phosphorus by the degradation of phytic acid in raw materials used for animal feed.

[0032] The phytases produced in the transgenic plants or seeds of the present invention can also be used in a process of dipping corn or sorghum kernels. The plant tissue or seed may be ground before addition to the steeped corn. Phytases released from plant tissues or seeds can act on phytin present in many corn preparations. The transgenic cereal grain (if it is a corn or sorghum grain) can produce phytase and degrade phytin in situ. Degradation of phytin in corn steep increases nutrient content and consequently increases the commercial value of corn steep liquor (used as animal feed or as a nutrient in microbial fermentation). In addition, the degradation of phytin may prevent problems with the accumulation of phytin deposits on filters, pipes, reactor vessels, etc. during concentration, transfer and storage of the corn steep liquor. The action of phytase may also facilitate the steeping and separation processes involved in corn wet milling.

Further, starch hydrolase (for example,
Amylopululanas
The use of transgenic seeds containing e)) may be compatible and desirable with the processes currently used in the food and wine industries for the production of sugars using cereal grain starch. Rice provides a major source of food for more than 50% of the world's population and is the most important and widely cultivated cereal on earth. Due to its large biomass, rice seeds are an ideal source of soluble sugars for the food and wine industries. Thermoanaerobactate, which possesses both pullulanase activity and α-amylase activity
Amylopullulanase (APU) from R. ethanolicus 39E is a polysaccharide α-1,4.
And both the α-1,6 bond can be hydrolyzed and are thermostable, with 90 ° C. being the optimal condition for the catalyst. Generally, rice contains 6-10% by weight protein and 70-80% by weight starch. APU can hydrolyze starch to produce soluble sugars and at the same time increase the relative composition of insoluble rice flour. High protein rice flour has high nutritional value and pudding,
Useful for producing porridge, instant milk, baby food, etc. As shown below, amino acids 75-1 of the mature APU of Ethanolics (total 1481 amino acids)
2.9 kb DNA of APU gene encoding 029
The fragment was isolated and characterized. This truncated APU maintains both α-amylase activity and pullulanase activity. In the production of sugar from seed starch, the use of transgenic rice seed containing dual active APU simplifies the production process and significantly reduces the production cost for rice flour.

The genetic elements essential for constructing a transgene to be inserted into a plant are readily available. For example, the α-amylase promoter is described in US Pat. No. 5,460,952 and the examples below. Other suitable promoters are described below:
cElroy et al., Plant Mol. Biol. 1
5: 257-268, 1990; Brusslan et al.,
Proc. Natl. Acad. Sci. USA 8
9: 7791-7795, 1992; Wissenba
ch et al., Plant J .; 4: 411-422,199
3; Reynolds et al., Plant Mol. Bio
l. 29: 885-896, 1995; Kaukine
n et al., Plant Mol. Biol. 30: 1115
１２８1128, 1996; Beaudoin et al., Plan.
t Mol. Biol. 33: 835-846,199
7; Moore et al., Proc. Natl. Acad. S
ci. USA 94: 762-767, 1997; Od.
ell et al., Nature 313: 810-812, 1
985; and Su-May Yu and Yu-Cha
nChao is listed as the inventor, 20
“Plant Seelin, filed May 22, 2000
US Patent Application entitled "g and Embryo Promotor". Other genetic elements (eg, cis regulatory sequences) are also available to one of skill in the art. See, for example, U.S. Patent No. 5,969,127.

The expression construct containing the transgene can be inserted into a vector (eg, a plasmid). This is then followed by Agroba, a standard method known in the art.
It is introduced into plants using cterium tumefaciens-mediated DNA delivery or microparticle bombardment.

The DNA sequence encoding the enzyme may be a microorganism,
It can be obtained from a variety of sources, such as plant or animal sources. For example, a DNA sequence encoding a phytase can be cloned from rumen bacteria, for example, by screening a bacterial cDNA library, or by PCR amplification of nucleic acids using heterologous or degenerate probes.

The cloned genes described herein can be used to construct "second generation" enzymes whose amino acid sequence has been altered by mutagenesis (eg, site-directed mutagenesis). Can be used as a starting material. This second generation enzyme has advantageous properties that are different from those of the wild-type or recombinant enzymes. For example, temperature or pH optimum, specific activity or substrate affinity can be altered to be more appropriate for application in a defined process.

Several techniques are available for introducing expression constructs containing enzyme-encoding DNA sequences into cells capable of producing plants or transgenic plants. Such techniques include electroporation, microinjection, sonication, polyethylene glycol-mediated protoplast transformation, poly-L-ornithine method, calcium phosphate method, and particle bombardment.

In addition to these so-called direct DNA transformation methods, a wide variety of transformation systems containing vectors such as viral vectors (eg, from cauliflower mosaic virus, from CaMV) and bacterial vectors (eg, from the genus Agrobacterium). Available.
After selection and / or screening, transformed protoplasts, cells or plant parts (eg, explants) can be regenerated into whole plants using methods known in the art.

For the transformation and regeneration of cereal crops,
One embodiment of the invention is a binary vector system (Hoe).
kema et al., Nature 303: 179-180,
1983; and European Patent Application No. 0120516). In this system, Agrobacterium
The m strain contains a vir plasmid having the virulence gene and a plasmid compatible with the gene construct to be transferred. As used in this example, the binary vector contains the hph gene encoding hygromycin resistance between the left and right border sequences of T-DNA,
And a multiple cloning site for insertion of the gene construct.

The promoter used to direct phytase expression may be the α-amylase gene promoter. The α-amylase gene promoter is used in germinating seeds of cereal crops (Huttly et al., EMBO).
J. 8: 1907-1913, 1989; Skrive
et al., Proc. Natl. Acad. Sci. USA
88: 7266-7270, 1991; Lanahan.
Et al., Plant Cell 4: 203-211, 19
92; Tanida et al., Mol. Gen. Genet.
244: 127-134, 1994; Itoh et al., Pl.
Ant Physiol. 107: 25-31,199
5), sucrose-deficient suspension cells (Chan et al., J. Bi
ol. Chem. 269: 17635-17641, 1
994), and other tissues and organs (Chan et al.,
Plant Mol. Biol. 22: 491-50
6, 1993).

The transgene may further comprise a signal peptide sequence derived from the α-amylase gene or another gene capable of directing the recombinant protein to the endoplasmic reticulum, vacuole, protein grain or extracellular space. Other regulatory sequences, such as a terminator sequence that functions in plants and a polyadenylation signal, can be included in the transgene. One example of one such sequence is Agroba
It is the 3 ′ untranslated region of the nopaline synthase (Nos) gene of C. tumefaciens or the 3 ′ untranslated region of the rice α-amylase gene (US Pat. No. 5,969,127).

Once the transgenic plants of the present invention have been produced, expression in tissues other than seed tissues can be beneficial. For example, a transgenic plant can be used as a producer of recombinant phytase, whether or not the phytase is isolated from the leaves, leaf sheaths, stems or roots of the transgenic plant.

[0044] Phytase activity can be measured using any method known in the art, including colorimetric techniques or native gel assays, including ELISAs, Western blots and direct enzyme assays. See, for example, Shimizu, Biosci. Biotech.
Biochem. 56: 1266-1269,199
2; Yanke et al., Microbiology 14.
4: 1565-1573, 1998; and Kim et al.
Enzyme Microb. Technol. 22:
2-7, 1998.

Transgenic plants, plant organs,
Alternatively, the malt seeds can be used directly (ie, without further processing) or first treated via conventional means (eg, grinding until uniform as appropriate for the particular use). Can be done. Alternatively, phytases can be extracted from plants, plant organs, or malt seeds and, if desired, can be purified prior to use using conventional extraction and purification techniques. For example, Labour
e et al., Biochem. J. 295: 413-419,
1993; and Golovan et al., Can. J. Mi
crobiol. 46: 59-71,2000.

Without further elaboration, it is believed that one skilled in the art can, based on the above disclosure, and the generation of transgenic plants and seeds described below, utilize the present invention to its fullest extent. The following examples are merely illustrative of the methods by which one skilled in the art may isolate and use the transgenic plants of the present invention, and should be construed as not limiting the remainder of the disclosure in any way. All publications, patents or patent applications cited in this disclosure are
It is incorporated herein by reference.

[0047]

EXAMPLES Example 1 The methods, materials, and procedures used in this example are first described.

(Methods and Materials) Plant materials. The rice variety used in this example was Oryza sativa.
L. cv. Taingung 67. The immature seeds are chaffed, sterilized with 2.4% NaOCl for 1 hour, thoroughly washed with sterile water, and washed with Toki, Plan
t Mol. biol. Rep. 15: 16-21, 1
Placed on N6D agar for callus induction as described in 997. One month later, callus from the scutellum was
% Sucrose and 10 μM 2,4-D,
Subcultured in fresh N6D medium or liquid MS medium for transformation, Yu et al. Biol. Che
m. 266: 21131-21137, 1991. Suspension cell cultures were established as previously described.

[0049] Plasmid. Plasmid αAmy8-C
Inserts a 1.4-kb rice α-amylase cDNA insert into pBluescript KS + (Stratatage).
ne) (Yu et al., Gene 122: 24)
7-253, 1992). Plasmid pRY18 contains 3.8 kb of DNA containing the rice genomic rDNA cluster (including the 3 'half of the 18S rRNA gene, the complete 5.8S rRNA gene, and the 5' half of the 25S rRNA gene). Fragment to p
In UC13 (Sano et al., Genome 3)
3: 209-218, 1990). Plasmid RAMY
G6a contains the 3 'half of the αAmy8 genomic DNA and the 3' flanking region. Plasmid RAMYG6
a to a rice genomic DNA library (Clontec)
h) was prepared by screening using αAmy8-C as a probe. Selected clones were subsequently purified from positive EMBL-3 phage clones into pBluescript (Stratagen).
Subcloned during e). Plasmid RAMYG
17 contains the 5 'flanking region, the entire coding region, and the 3' flanking region of αAmy7 genomic DNA. Plasmid R
AMYG17 was converted to a rice genomic DNA library (Cl
onte) was screened using αAmy8-C as a probe. RAMYG6
a and further details on RAMYG17 can be found in Ho
Et al., Master's thesis, Department of Biol
ogy, National Taiwan Norma
l University, 1991.
The selected clones were subsequently cloned into a positive EMBL
-Plasmid construction subcloned into pBluescript from a phage clone. S. rumin
The 5 'end of the antium phytase gene (SrPf6) was used as a DNA template for plasmid pSrPf
6 (US Pat. No. 5,939,303) and a primer

[0050]

Embedded image (SEQ ID NO: 1; EcoRV site is underlined) and 5'-ACGCA GGATCC ACCTCATAAAAA
Modified by PCR using CC-3 '(SEQ ID NO: 2; BamHI site is underlined) so that the EcoRV site identifies the first amino acid of the mature, processed phytase enzyme (SEQ ID NO: 1). (Bold) integrated 5 'to BamH
A recombinant gene having an I site at the end of this phytase gene was obtained. The EcoRV site allows fusion of the phytase gene to the signal peptide sequence of the α-amylase gene αAmy8. The PCR product is
Ec from Bluescript (Stratagene)
Subcloning into the oRV and BamHI sites generated pBS / SrPf6.

E. The E. coli phosphatase gene appA is transformed into a plasmid p as a DNA template.
ET-appA (Golovan et al., Can. J. Mi.
crobiol. 46: 59-71, 2000), and oligonucleotide 5'-ACGGCG GATAT
C CAGAGTGAGCCGGAGCTTGA-3 ′ (SEQ ID NO: 3 EcoRV site is underlined) and 5 ′
-AGGTTT GGATCC TTACAAACTGCAC
Similarly, 5 ′ was obtained by PCR using GAAGG GT-3 ′ (SEQ ID NO: 4; BamHI site is underlined).
Modified at the end and 3 'end. The resulting PCR product was subcloned into pBluescript,
S / appA was generated.

The 1.2 kb promoter and signal peptide regions of αAmy8 were replaced with SalI and Hind.
III and pAG8 (Chan et al., Plant Mol.
Biol. 22: 491-506, 1993) and subcloned into pBluescript to generate pBS / 8SP. Using the 3 ′ untranslated region (3 ′ UTR) of αAmy8 as a DNA template, R
AMYG6a and oligonucleotide 5′-CG
GGATCC TAGCTTTAGCTATAGCGAT
-3 '(SEQ ID NO: 5; BamHI site is underlined)
And 5'-TCC CCGCGG GTCCTCTAAG
PCR amplification was performed using TGAACCGT-3 ′ (SEQ ID NO: 6; SacII is underlined). Nopaline synthase gene (Nos) 3'UTR, pBI221 (Clontech) as DNA template, and oligonucleotide 5'-TCC GGATCC CAGA
TCGTTCAAAACATTT-3 ′ (SEQ ID NO: 7; B
amHI site is underlined) and 5'-AGC CC
GCGG GATCGATCTAGTAACAT-3 '
(SEQ ID NO: 8; SacII is underlined)
CR amplified.

ΑAmy8 and Nos3′UTR were replaced with p
Subcloning into the BamHI and SacII sites in BS / 8SP, pBS / 8SP8
U and pBS / 8SPNos were generated. SrPf6
With pBS / Sr using EcoRV and BamHI
Excised from Pf6 and subcloned into the same sites in pBS / 8SP8U and pBS / 8SPNos to obtain pBS / 8F8U and pBS / 8F, respectively.
N was generated. The appA gene was transformed with EcoRV and B
It was excised from pBS / appA using amHI and subcloned into the same site in pBS / 8SP8U and pBS / 8SPNos to generate pBS / 8A8U and pBS / 8AN. Appropriate in-frame fusion of the αAmy8 signal peptide sequence to the SrPf6 or appA coding region, and the SrPf6 or appA coding region to the αAmy8 3′UTR or N
All joining regions linked to the os 3'UTR were confirmed by DNA sequencing.

The 1.7-kb promoter and signal peptide regions of αAmy7, RAMYG17a as a DNA template, and oligonucleotide 5′-ACCGG GTCGAC GTATACATGTC
ACCTACA-3 '(SEQ ID NO: 9; SalI site is underlined) and 5'-GGT GATATC CAGG
ACTT GCCCGGCTGT-3 ′ (SEQ ID NO: 1
0; the EcoRV site is underlined) and the SalI site is 5 ′ of the αAmy7 promoter.
A PCR product was obtained with a terminus and an EcoRV site immediately 3 'to this signal peptide sequence. This PC
The R product was subcloned into the SalI and EcoRV sites in pBluescript to generate pB
S / 7SP was generated. αAmy7 3′UTR is D
RAMYG17 as NA template and oligonucleotide 5'-GC TCTAGA AATCTGA
GCGCACGATG-3 ′ (SEQ ID NO: 11; XbaI
Sites are underlined) and 5'-TCC CCGCGG
PC using TAAGCATTAAGCAGTGCA-3 '(SEQ ID NO: 12; SacII site is underlined)
R amplification was performed. This PCR product was cloned into XBS in pBS / 7SP.
Subcloning into the baI and SacII sites generated pBS / 7SP7U. Nos 3'UT
R was converted to pBS / 8S using XbaI and SacII.
It was excised from PN and subcloned into the same site in pBS / 7SP to generate pBS / 7SPNos. The SrPf6 gene was ligated with EcoRV and BamHI.
Is excised from pBS / SrPf6 using
Subcloned into the same site in s / 7SP7U and pBS / 7SPNos, respectively, pBS / 7F7
U and pBS / 7FN were generated. The appA gene was transformed into pBS / ap using EcoRV and BamHI.
excised from pA and pBS / 7SP7U and p
Subcloned into the same site in BS / 7SPNos, pBS / 7A7U and pBS / 7AN, respectively.
Generated. SrP of αAmy7 signal peptide sequence
The appropriate in-frame fusion with the f6 or appA coding region, as well as the joining region linking the SrPf6 or appA coding region to the αAmy7 3′UTR or Nos 3′UTR, were all confirmed by DNA sequencing.

Cauliflower mosaic virus 35SR
NA (CaMV35S) promoter-Hygromycin B phosphotransferase (hph) coding sequence-
The tumor morphology large gene 3 ′ UTR (tml) fusion gene was ligated to pTRA151 (Zheng
Et al., Plant Physiol. 97: 832-83
5, 1991) and the binary vector pPZP200 (Hajdukiewicz et al., Pl.
ant Mol. Biol. 25: 989-994, 1
994) by subcloning into the EcoRI site.
PZP / HPH was produced. pPZP / HPH, Sa
Linearized with I1 and blunt-ended to generate a vector for cloning. pBS / 8F
8U, pBS / 8FN, pBS / 8A8U, pBS / 8
AN, pBS / 7F7U, pBS / 7FN, pBS / 7
A7U and pBS / 7AN were linearized with PvuII and used as inserts for cloning.
This insert was ligated with the vector to give pPZP /
8F8U, pPZP / 8FN, pPZP / 8A8U, p
PZP / 8AN, pPZP / 7F7U, pPZP / 7F
N, pPZP / 7A7U, and pPZP / 7AN were generated.

Transformation of rice. Plasmid was transferred to Agro
bacterium tumefaciens strain EHA
101 (Hood et al., J. Bacteriol. 16).
8: 1291-1301, 1986) using an electroporator (BTX) according to the manufacturer's instructions. Callus derived from immature rice seeds,
Hiei et al., Plant J. 6: 271-282, 1
Co-culture with Agrobacterium according to the method described by 994 and Toki et al., Supra.

Northern blot analysis. The total RNA was
Et al., Plant Mol. Biol. 30: 1277-
1289, 1996 and isolated from endosperm of germinating seeds and TRIZOL reagent (GIBCO
BRL) from cultured suspension cells. RNA
Gel blot analysis was performed using the method of Thomas, Methods E.
nzymol. 100: 255-266, 1983. Briefly, 10 μg of total RNA
Was electrophoresed on a 1% agarose gel containing 10 mM sodium phosphate buffer (pH 6.5), transferred to a nylon filter, and labeled with ³² P random primer, SrPf6 DNA, appA DNA, αAmy.
Hybridized with 8 gene-specific DNA or rDNA probes. The αAmy8 gene-specific DNA was
Heu et al. Biol. Chem. 271: 2699
Prepared as described in 8-27004, 1996. Blots were visualized using autoradiography and PhosphoImager (Molec
Quantitation was performed using the software accompanying (Ultra Dynamics).

Western blot analysis. Total protein was extracted with extraction buffer (50 mM Tris-HCl [pH
8.8], 1 mM EDTA, 10% glycerol,
Extracted from endosperm of germinating seeds using 1% Triton X-100, 10 mM β-mercaptoethanol, and 0.1% sarkosyl). Western blot analysis was performed according to Yu et al. Biol. Chem. 266:
21131-21137, 1991.

Phytase activity assay. The protein extract was prepared according to Li et al., Plant Physiol. 114:
Prepared from germinating seeds as described in 1103-1111, 1997. Phytase activity is measured by Shimi
zu, Biosci. Biotech. Bioche
m. 56: 1266-1269, 1992.

(Results) The αAmy8 promoter was expressed in appA and SrP in transformed rice suspension cells.
Confers enhanced sucrose starvation accumulation of f6 mRNA.
To determine whether the αAmy8 promoter regulates sugar-dependent expression of appA in rice, αAm8
A 1.2-kb DNA fragment containing the 5 'regulatory sequence of y8 and the signal peptide sequence was fused in frame at the 5' end of the appA or SrPf6 gene. This chimeric gene was inserted into a binary vector and used for Agrobacterium transformation for rice transformation.
rium. Many transformed cell lines were obtained and eight were selected for further study. The transformed calli were cultured as suspension cells, which were then cultured in sucrose-free or sucrose-free medium. Total RNA is purified and ap
Gel blot analysis was performed using pA DNA, SrPf6 DNA, αAmy8 gene-specific DNA, or rDNA as a probe. Accumulation of appA, SrPf6, and αAmy8 mRNA was detectable in sucrose-starved cells but not in cells that provided sucrose. appA mR
Neither NA nor SrPf6 mRNA was detected in non-transformed (NT) cells.

The αAmy8 promoter confers sucrose starvation enhancing activity of phytase in transformed rice suspension cells. αAmy8-appA or αAm
To determine whether transformed rice suspension cells carrying the y8-SrPf6 chimeric gene synthesize phytase and secrete it into the culture medium, determine the phytase activity in the suspension cells and in the culture medium. It was determined. Phytase activity was detectable in transformed cells and culture media. In both cases, phytase activity was significantly enhanced by sucrose starvation. Phytase activity was also detectable in untransformed cells under sucrose starvation. This was probably caused by the endogenous phytase gene.

The αAmy7 and αAmy8 promoters are responsible for Sr in germinating transgenic rice seeds.
Controls expression of Pf6. Transgenic rice plants were transformed with αAmy7-SrPf6 or αAmy8-SrP
Regenerated from transformed rice calli carrying f6.
T1 seeds of several transgenic lines were randomly selected for further study. Transgenic rice seeds were germinated for 5 days to determine the role of the αAmy7 and αAmy8 promoters in the expression of the appA and SrPf6 genes during seed germination. Total RNA was purified from whole germinated seeds (including shoots, roots, and endosperm) and SrPf6 DNA, αAmy8 gene specific DN
A, or subjected to gel blot analysis using rDNA as a probe. Under the control of the αAmy7 promoter,
SrPf6 expression can be detected in some of the germinated seeds. Under the control of the αAmy8 promoter, S
rPf6 expression can be detected in all of the germinated seeds. SrPf6 mRNA was not detected in non-transformants.

The αAmy8 promoter controls appA expression in germinating transgenic rice seeds. Transgenic rice plants were transformed with αAmy8-a
Regenerated from rice calli carrying ppA. T1 seeds of several transgenic lines were randomly selected for further study. Seeds are allowed to germinate for 5 days and total RNA is transferred to whole germinated seeds (shoot,
(Including roots and endosperm) and appA
DNA, αAmy8 gene-specific DNA, or rDN
A was subjected to gel blot analysis using as a probe.
Under the control of the αAmy8 promoter, expression of appA can be detected in all germinated seeds. app
A mRNA was not detected in non-transformants.

The αAmy8 promoter confers high phytase activity in germinating transgenic rice seeds. Phytase activity in T1 transgenic seeds was analyzed to determine the level of phytase expression in germinated transgenic seeds. Transgenic rice seeds were germinated for 5 days. Cell extracts were prepared from whole germinated seeds (including shoots, roots, and endosperm) and subjected to phytase activity analysis. Phytase activity was determined by αAmy8-SrPf6-N
It was higher in all germinated transgenic seeds carrying the os chimeric gene than in non-transformants.
One of the transgenic lines, 8FN-8, had significantly higher phytase activity compared to the other transgenic lines. The phytase activity was αAmy8
In all germinated transgenic seeds carrying the -appA chimera gene, it was higher than in non-transformants. One transgenic line, 8AN-1
4 had significantly higher phytase activity compared to other transgenic lines containing 8FN-8.

The introduced phytase and acid phosphatase genes are inherited in progeny of transgenic rice. Introduced appA DNA and SrPf6
To determine whether the DNA was inherited in T1 transgenic rice progeny, transgenic line 8A
N-14 and 8FN-8 T2 seeds were germinated for 5 days. Total RNA was purified from whole germinated seeds and analyzed for appA DNA, SrPf6 DNA, or rD.
The samples were subjected to gel blot analysis using NA as a probe. The expression of appA and SrPf6 is dependent on the germinated T2
It was detected in most of the seeds.

The αAmy8 promoter confers transient expression of phytase in germinating transgenic rice seeds. αAmy8-appA or αAmy8
To determine the expression pattern of phytase in germinated transgenic rice seeds carrying SrPf6, transgenic lines 8FN-8-8 and 8
AN-14-10 T2 seeds were germinated for various periods of time and phytase activity was determined. Phytase activity in transgenic seeds increased as germination progressed, peaked on days 5-6, and decreased thereafter. Phytase activity was also detectable in untransformed seeds, but remained low throughout the germination period.

The molecular weight of phytase produced in germinating transgenic rice seeds was determined by Pichia
similar to the molecular weight of phytase produced in P. pastoris. SrPf6 was converted to glyceraldehyde-3
-Cloned into a plasmid under the control of the phosphate dehydrogenase (GAP) promoter and
ia pastoris. SrPf
6 Using cDNA as pSfPf
6 and oligonucleotide 5′-CG GAATTC GCCAAGGCCGCCGG as 5 ′ primer
AGCAGAC-3 '(EcoRI site is underlined), and 5'-GC TCT as 3' primer
AGA TAGCCCTTCGCCGGATGGCT-
PCR amplification was performed using 3 '(XbaI site is underlined). The DNA fragment containing the SrPf6 cDNA was digested with EcoRI and XbaI and
Ligation at the same site in GAPZaA (Invitrogen) generated pGAP-SrPf6. SrPf
6 followed the signal peptide α-factor and was under the control of the GAP promoter. pGAP-SrPf6
Was linearized with the restriction enzyme AvrII, and P. by electroporation. pastoris host strain SMD1168H. The transformed cells were transformed with YPDS (1% yeast extract, 2% peptone, 2%
Dextrose, 1M sorbitol, pH 7.5)
Plated at 30 ° C. for 3 days. Phytase production and purification were performed according to the manufacturer's instructions provided with pGAPZaA.

To compare the molecular weight of phytase produced in germinating transgenic rice seeds with the molecular weight of phytase overexpressed in Pichia, seeds of transgenic rice line 8FN-8 were germinated for 5 days. . Total protein was extracted from whole germinated seeds and subjected to Western blot analysis using phytase polyclonal antibodies. SrPf6
Full-length cDNA, plasmid pSrPf6 as a DNA template, and oligonucleotide 5′-CCC GAATTC ATGAAA as a 5 ′ primer
TACTGGCAG-3 '(EcoRI site is underlined) and 5'-CCC GA as 3' primer
GCTC TTACGCCTTCGCCGGG-3 '(Sa
(The cI site is underlined). The amplified DNA fragment was subjected to EcoRI and Sac
I and digested with pET20b (+) (Novage
Ligation to the same site in n) generated pET-SrPf6. pET-SrPf6 was purchased from E.I. coli strain BL2
1 (DE3) and expressed. Purification of the phytase was performed according to the instructions provided by Novagen. One hundred micrograms of purified phytase was obtained from Williams et al., "Expressi.
on of foreign proteins in
E. FIG. colising plasmid vector
ors and purification of s
peculiar polyclonal antibo
dies ", DNA Cloning Expre
session Systems: A Practical
Approach, Glover et al., IRL Pr.
New Zealand White rabbits were injected continuously at 4-6 week intervals according to the method generally described in ess, New York, 1995.

The molecular weight of full-length phytase produced by germinating transgenic seeds was similar to the molecular weight of phytase produced by Pichia.
The phytase encoded by SrPf6 is E. coli. c
It was known to have a molecular weight of 36.5 kD when expressed in oli (US Pat. No. 5,939,3).
03). The molecular weight of the phytase expressed in Pichia was about 36.5 kD and about 38 kD. Similarly, when phytase was expressed in transgenic rice seed, the molecular weight was about 36.5 kD and about 40 kD. In the primary structure of SrPf6, 1
It was known that there were two potential glycosylation sites (US Pat. No. 5,939,303). The 38 kD and 40 kD phytases expressed in Pichia and transgenic rice seeds, respectively, were probably caused by different glycosylation. One protein with a molecular weight of 34 kD was also present in germinating rice seeds, which could be a degradation product of phytase. These results indicate that the mature phytase produced in the transgenic plants and seeds described herein was appropriately post-translationally modified.

Transgenic germinated rice seeds /
Seedlings have a wide range of pH for high specific activity and high activity
It contained a phytase with a profile. Surprisingly, the specific activity of phytase expressed in germinated transgenic seeds was higher than that of E. coli. It was also found to be twice as high as phytase expressed in E. coli. The reason for this surprising result may be that there are many endogenous hydrolases that are simultaneously expressed in germinated seeds. These hydrolases may have a synergistic effect on phytase activity present in germinating seeds. Alternatively, or in conjunction, post-translationally modified phytase in germinating seeds may have increased specific activity of the enzyme, which may result in the observed increase in phytase activity. Thus, in addition to the advantage of expressing phytase in transgenic malted seeds, the specific activity of the recombinantly produced enzyme was also increased.

Ruminal bacterial phytase has an optimum pH of 4.0 to 5.5 (US Pat. No. 5,939,303). To determine the optimal pH of phytase expressed in rice seeds above, T2 seeds of three transgenic rice lines were germinated for 5 days and the optimal pH for phytase expressed in rice germinated seeds / seedlings It was determined. The results show that the activity of phytase expressed in all three transgenic rice lines was pH3 and pH3.
It was shown to be optimal at 4.5 to 5.0 (FIG. 1).
In the gastrointestinal tract of monogastric animals, the pH is 2-3 in the stomach,
And in the small intestine it is in the range of 4-7. The broader and more acidic pH profile of the phytase expressed in rice allows this enzyme to function well in the stomach and small intestine of animals, and therefore as a feed additive Advantageous for its use. The reason for this surprising result may be that the phytase expressed in rice was post-translationally modified. Post-translational modifications have resulted in increased activity and / or stability over a wide range of pH.

E. E. coli acid phosphatase
With an optimum pH of 2.5 (Dassa et al., J. Bio
l. Chem. 257: 6669-6676, 198.
2; and Dassa et al. Bacteriol. 1
72: 5497-5500, 1990). To determine the optimal pH of acid phosphatase expressed in rice, T2 seeds of transgenic rice line 8AN-14-6 were germinated for 5 days and the optimal pH of phytase expressed in germinated rice seeds / seedlings It was determined. The results show that the activity of acid phosphatase expressed in rice was optimal over a pH range of 3-5.5 (FIG. 2). As for ruminal bacterial phytase expressed in rice, c
The broader and more acidic pH profile of oli acid phosphatase allows this enzyme to function well in the stomach and small intestine of animals, and is therefore advantageous for its use as a feed additive. is there.
The reason for this surprising result could also be that the acid phosphatase expressed in rice was post-translationally modified. Post-translational modifications have resulted in increased activity and / or stability over a wide range of pH.

Example 2 First, the method, materials and procedures used in this example will be described.

(Materials and Methods) Plant materials. All plant materials were prepared as described in Example 1.

Preparation of genomic DNA. Rice seeds were germinated and grown in the dark for one week. Bacteria T. etha
nolicus 39E was obtained from the American Type Culture Collection (ATCC 53033). This bacterial and rice genomic DNA was prepared as described by Sheu et al. B
iol. Chem. 271: 26998 to 27004,
Purified according to the method described in 1996.

PCR. 1351 bp glutelin gene promoter region (fragment I; Zheng et al., P
lant J. 4: 357-366, 1993; and Wu et al., Plant Cell Physiol. 3
9: 885-889, 1998), using rice genomic DNA as a template, and primer B1-5 (5 ′
-GGG GAATTC GATCTCGATTTTGA
GGAAT-3 ′ (SEQ ID NO: 13), EcoRI sites are underlined), and B1-3 (5′-GGG GGAT)
CC CATAGCTATTTGTACTTGCT-3 '
(SEQ ID NO: 14), the BamHI site is underlined). This glutelin gene promoter and a 75 bp putative signal peptide sequence (fragment II) were used as a template for rice genome DN.
A, primer B1-5, and primer B1-sp
(5'-GGG GGATCC GGGATTAAATAG
CTGGGCCA-3 ′ (SEQ ID NO: 15), BamHI
(The site is underlined). This glutelin gene promoter and the putative signal peptide and propeptide sequences (fragment III) were used as templates with rice genomic DNA, and primers B1-5 and B1-pro (5′-GGG GGAT).
CC CCTCACTTTCCGAAGTGGTTT-3 '
(SEQ ID NO: 16), the BamHI site is underlined).

A truncated apu ORF encoding only amino acids 75 to 1029 was used as a template in T. cerevisiae. et
hanolicus 39E genomic DNA, and oligonucleotide 5′-CG G as a forward primer
GATCC TTAAGCTTGCATCTTGA-3 '
(SEQ ID NO: 17; BamHI site is underlined), and 5′-CCG GCGGCCG as reverse primer
C CTACATAATTTTCCCCTTGGCCA-
PCR amplification was performed using 3 ′ (SEQ ID NO: 18; NotI site is underlined).

Plasmid construction. The PCR amplified fragments I, II, and III were digested with EcoRI and BamHI and pBluescript
(Stratagene), subcloned into the same site, pBS-G, pBS-Gp, and pBS-G, respectively.
BS-Gpp was generated. This shortened type apu is called Bam
Digested with HI and NotI, and pBS-G, pB
Fused to the GluB-1 promoter, the GluB-1 promoter-signal peptide sequence, and the GluB-1 promoter-signal peptide-propeptide sequence in S-Gp and pBS-Gpp, respectively.
The translation fusions were made and each plasmid pBS
-G-apu, pBS-Gp-apu, and pBS-
Gpp-apu was prepared. The 3 'untranslated region (UTR) of the nopaline synthase gene (Nos) was ligated with pBI221 (Clontech) as a DNA template and oligonucleotide 5'- as a forward primer.
TCC GAGCTC CAGATCGTTCAACACAT
TT-3 '(SEQ ID NO: 19; SacI site is underlined), and oligonucleotide 5'-AGC GAGCTC GATCGATCTAGGT as reverse primer
PCR amplification was performed using the AACAT-3 ′ (SEQ ID NO: 20; SacI site is underlined). This Nos
The 3'UTR is digested with ScaI and pBS-G-
apu, pBS-Gp-apu, and pBS-Gpp
-Fused downstream of apu in apu, each with pBS
-G-apu-Nos, pBS-Gp-apu-No
s and pBS-Gpp-apu-Nos.

ΑAmy8 (Chan et al., Plant M
ol. Biol. 22: 491-506, 1993) was excised from pAG8 with SalI and HindIII and subcloned into pBluescript to generate pBS / 8sp. 3'U of αAmy8
TR was replaced with RAMYG6a as a DNA template and oligonucleotide 5'- as a forward primer.
CG CCGCGG TAGCTTTAGCATATAGCG
AT-3 '(SEQ ID NO: 21; SacII site is underlined), and oligonucleotide 5'-TCC CCGCGCGTCCTCTAAGTG as reverse primer
PCR amplification was performed using AACCGT-3 ′ (SEQ ID NO: 22; SacII site is underlined). Plasmid R
AMYG6a contains the 3 'half and the 3' flanking region of the coding sequence of αAmy8 genomic DNA. This plasmid was used as a probe with αAmy8-C (Yu et al., Gen.
e 122: 247-253, 1992).
It was generated by screening a rice genomic DNA library (Clontech). This α Am
The 3'UTR of y8 was subcloned into the SacII site in pBS / 8sp to generate pBS / 8sp8U. Truncated apu was cut with BamHI and NotI and subcloned into the same site in pBS-8sp8U to generate pBS-αAmy8-sp-apu-8U.

The 1.1 kb promoter and signal peptide sequence of αAmy3 was replaced with p3G-132II (Lu
J. et al. Biol. Chem. 273: 10120-1
0131, 1998) from SalI and HindII.
I and excised into pBluescript to generate pBS-3sp. This α Am
The 3'UTR of y3 was converted to pMTC37 (Chan et al., Pl.
ant J. 15: 685-696, 1998) to H
Excision with indIII and SacI and pBS
Subcloning into the same site in -3sp to give pBS-3
sp3U was generated. The truncated apu was digested with BamHI and NotI and subcloned into the same site in pBS-3sp3U to obtain pBS-αAmy3-sp.
-Apu-3U was generated.

GluB, αAmy3 and αAmy8
Of the signal peptide or propeptide sequence of
Precise in-frame fusion with coding region, αAmy3,
The binding region linking the αAmy8 or Nos 3′UTR to the apu coding region was all confirmed by DNA sequencing. This GluB-apu-Nos chimeric gene, GluB-sp-apu-Nos chimeric gene, GluB-spp-apu-Nos chimeric gene,
αAmy3-sp-apu-αAmy3 3′UTR chimeric gene, and αAmy8-sp-apu-αAm
The y8 3'UTR chimeric gene was transformed into p
BS-G-apu-Nos, pBS-Gp-apu-N
os, pBS-Gpp-apu-Nos, pBS-αA
my3-sp-apu-3U, and pBS-αAmy
Excised from 8-sp-apu-8U, blunt-ended,
The HindIII digested and blunt-ended binary vector pSMY1H (Ho et al., Plant Ph.
ysiol. 122: 57-66, 2000), respectively, pSMY1 / Gapu, pSMY1 / Gp
apu, pSMY1 / Gppappu, and pSMY1
/ 3apu and pSMY1 / 8apu were produced.

Transformation. Transformation was performed as described in Example 1.

E. Expression of APU in E. coli and preparation of antibodies. Truncated apu encoding amino acids 75 to 1029 was used as a template for T. cerevisiae. etanolic
us39E genomic DNA and oligonucleotide 5'-CG CATATATG TTA as forward primer
AGCTTGCATCTTGATTC-3 '(SEQ ID NO: 23; underlined NdeI site), and 5'-CCG CTCGAG CTACATA as reverse primer
TTTTCCCCTTGGCCA-3 ′ (SEQ ID NO: 2
4; XhoI site is underlined). The amplified DNA fragment was digested with NdeI and XhoI and pET20b (+) (N
ovagen) to generate pET-APU. pET-APU was purchased from E.I. coli BL21
Strain (DE3) was transferred and APU was expressed. A
Purification of the PU was performed according to the instructions provided by Novagen. 100 micrograms of purified A
PUs were collected at 4-6 week intervals according to the method described in Williams et al., Supra, New Zealand.
d White rabbits were injected continuously.

APU activity assay and enzyme-linked immunosorbent assay (ELISA). Rice seed or tissue,
Triturate in liquid N ₂ and buffer (90.8 mM K ₂ HPO)
₄ , 9.2 mM KH ₂ PO ₄ , 10 mM EDTA, 1
0% glycerol, 1% Triton X-100,
And 7 mM β-mercaptoethanol) and centrifuged at 15,000 × g for 10 minutes. The supernatant was then collected. APU activity, Math
upala et al. Biol. Chem. 268: 16
332-16344, 1993. The ELISA was performed according to Ausubel et al., Shor.
t Protocols in Molecular
Biology, 2nd edition, A Compendium
of Methods from Current P
rotocols in Molecular Bio
logy, John Wiley & Sons, Ne
Performed as described in w York, 1992. Total protein concentration was determined using a Bradford dye binding assay (Bradford, Anal. Bioche).
m. 72: 248-254, 1976).
-Determined using the Rad protein assay kit.

Determination of sugar concentration. Mature rice seeds were ground to a powder using a mortar and pestle. The rice seedlings, triturated in liquid N ₂ using a pestle and mortar, to a powder.
Water was added to this powder to make a 10% (w / v) slurry. The slurry from this mature rice seed was heated at 90 ° C. for 30 minutes to extract total soluble sugars and
n et al., Plant J .; 6: 625-636, 1994
The sugar concentration was determined as described in. The slurry from rice seedlings was heated at 85 ° C. for 2 hours to extract total soluble sugars and the concentration of glucose, fructose, sucrose, maltose, and maltotriose to S
Haw et al., Biosci. Biotech. Bioch
em. 56: 1071-1073, 1992 as described in High Performance Liquid Chromatography (HPL
C).

(Results) Isolation of the promoter sequence, signal peptide sequence and propeptide sequence of the rice glutelin gene. Glutelin gene (GluB-1) (Ta
Kaiwa et al., Plant Mol. Biol. 17:
875-885, 1991), and three DNA fragments containing the GluB-1 promoter, putative signal peptide sequence, and putative propeptide sequence were synthesized using PCR. The putative 25 amino acid signal peptide cleavage site was determined by statistical methods (von Heijne,
J. Mol. Biol. 184: 99-105, 198
Estimated based on 5). A putative 36 amino acid propeptide cleavage site was identified as sweetpotato sporamine
in) propeptide sequence (Matsuoka et al., Pr.
oc. Natl. Acad. Sci. USA 88:83
4-838, 1991). Fragment I has 1351b ending at the right of the translation initiation codon.
pGluB-1 promoter region. Fragment II contained Fragment I with the addition of a 75 bp signal peptide sequence. Fragment II
I contained fragment II with a 108 bp deduced propeptide sequence. The purpose of including this signal or propeptide region was to target APU to different cellular compartments (eg, cytoplasm, protein body, or vacuole). Then, A in the transgenic rice seed
The effects of cellular localization on PU production, stability, and activity can be compared. Transformed rice suspension cells expressed and secreted APU in the culture medium.

The transformed rice suspension cells expressed and secreted APU in the culture medium. All plasmids were delivered to rice calli by biolistic or Agrobacterium-mediated transformation systems. Transfected rice calli were grown under hygromycin selection. Genomic Southern blot analysis of the transformed cell lines generally revealed that Agrobacterium
When the transgenic strain obtained via m-mediated transformation contains 1-2 copies of the apu gene in the genome, and via microprojectile bombardment, the transgenic plant has multiple copies (more than 2). The apu gene was found to be included in the genome.

The transformed rice calli were cultured in liquid MS medium to generate a suspension cell culture. APU is expressed with a signal or propeptide sequence in most transgenic strains,
Culture media was collected for analysis of APU accumulation. The amount of APU in the medium of the transformed rice suspension cell culture is
ELISA and purified as standard. coli expression AP
It measured using U. As shown in FIG. 3, the amount of APU in the medium of the transformed suspension cells was significantly higher than the amount of APU in the medium of the non-transformed cells. This figure 3
In, the black bar indicates a plant sample grown on sucrose. White bars indicate plant samples grown without sucrose. "NT" is a non-transformed control. In the medium of cells carrying the αAmy3 or αAmy8 promoter, the amount of APU was higher in the absence of sucrose than in the presence of sucrose.

Surprisingly, while the GluB-1 promoter has been shown to confer endosperm-specific expression in developing rice seeds, APU is also expressed in cultured rice suspension cells. Was found. GluB
AP in the medium of cells carrying the -1 promoter
The amount of U was higher in the presence of sucrose than in the absence of sucrose. In addition, APU expressed without the signal peptide sequence was also present in the culture medium. Computer analysis shows that the N-terminus of APU is
It was shown to have amino acid sequence characteristics similar to the signal peptide sequence. This may explain that APU is secreted into the culture medium without the addition of the leader peptide. Thus, it was discovered that APU without the signal peptide sequence could be secreted into the culture medium.

Transgenic germinated rice seeds /
Seedlings contain a high level of APU. Transgenic calli carrying apu under the control of the α-amylase gene promoter or the glutelin gene promoter and with or without a signal peptide sequence were regenerated. These transgenic plants are
Self-fertilized for generations and obtained T2 homozygous seeds.

The homozygosity of the transgenic seed was
25 transgenic seeds were germinated for 7 days in water containing 50 μ / ml hygromycin and then determined by calculating the ratio between the number of growing and non-growing seeds. Homozygous seeds were expected to germinate and grow under hygromycin treatment.
5 T2 homozygous seeds from each transgenic rice line carrying the various constructs were germinated and 5
Grow for days. Collect the fully germinated seeds / seedlings,
APU levels in cell extracts were then determined by ELISA and purified E. coli as standard. It was measured using E. coli-expressed APU. The average APU level expressed under the control of either the α-amylase gene promoter or the glutelin gene promoter was higher than that of non-transformants (FIG. 4). In FIG. 4, “NT” is a non-transformed control. The expression of APU in germinated rice seeds / seedlings was unexpected. Because Gl
This is because the uB-1 promoter was known to be specifically expressed in endosperm during seed development.

Transgenic mature rice seeds contain high levels of APU. Five T2 homozygous seeds from each transgenic rice line carrying the transgene were selected for APU analysis. Embryos and endosperm of mature seeds were collected individually and homogenized in buffer to form extracts. The APU level of the cell extract was determined by EL
It was measured using ISA. The average APU levels expressed in transgenic seeds under the control of either the α-amylase gene promoter or the glutelin gene promoter were higher than in non-transformed seeds (FIG. 5). In FIG. 5, "NT"
Is a non-transformed control. Unexpectedly, the GluB-1 promoter conferred APU expression in embryos. Furthermore, the fact that the αAmy3 and αAmy8 promoters conferred APU expression in embryos and endosperm of mature seeds was also unexpected. This is because the α-amylase gene was known to be specifically expressed in endosperm or embryos of transiently germinating seeds and not in mature seeds.

Transgenic germinated rice seeds /
Seedlings contain APU with high specific activity. The data in FIG. 4 showed that APU levels were significantly higher in transgenic germinated rice seeds / seedlings than in untransformed controls. To compare the specific activity of recombinant APU expressed in transgenic germinated seeds / seedlings, five T2 homozygous seeds of each transgenic rice line carrying various constructs were germinated and left for 5 days Grew. A fully germinated seed / seedling cell extract was prepared and APU levels in the cell extract were measured using ELISA. APU per APU amount in cell extract
Activity was measured after incubating the cell extract at 90 ° C. for 30 minutes. As shown in FIG.
A present in transgenic germinated seeds / seedlings
The specific activity of PU varied between lines, regardless of the construct used. Interestingly, the specific activity of APU expressed in transgenic germinated seeds / seedlings was higher in E. coli. than the activity of APU expressed in E. coli
7 times higher. The reason for this surprising result may be the presence of many endogenous hydrolases in rice endosperm. These hydrolases may have a synergistic effect on the APU activity present in germinated seeds. Alternatively, in combination, post-translationally modified APU in germinated seeds may increase the specific activity of the enzyme, which may cause the observed increase in APU activity. Note that there are three potential glycosylation sites in APU polypeptides, which can be glycosylated differently depending on the production method used. Thus, in addition to the above advantages of APU expressed in transgenic germinated seeds, the specific activity of the recombinant enzyme can also be increased.

Transgenic rice seeds and germinated seeds / seedlings produce high levels of sugar. The results summarized in FIGS. 4 and 5 revealed that APU levels were significantly higher in transgenic mature and germinated seed / seedlings than in untransformed controls. To determine whether transgenic seeds produce higher levels of soluble sugars than untransformed, five T2s of each transgenic rice line carrying apu under the control of the GluB-1 promoter. Homozygous seeds were homogenized in water and incubated at 85 ° C for 2 hours. As shown in FIG. 7, the concentration of soluble sugar in the transgenic seed was higher than in the non-transformed seed. In FIG. 7, “NT” is a non-transformed control. Five T2 homozygous seeds of each transgenic rice line carrying apu under the control of the αAmy3 and αAmy8 promoters were also homogenized in water, and
Incubated at 0 ° C. for 30 minutes. As shown in FIG. 8, the concentration of sugar in germinated seeds / seedlings was higher than in the untransformed control. FIG.
, "NT" is a non-transformed control. These results demonstrated that increasing APU levels in transgenic or germinated seeds / seedlings promotes sugar production from seed starch without the addition of exogenous APU.

[0095]

Thus, the present invention provides a transgenic plant that produces seeds useful as a feedstock for phosphorus or carbohydrate rich feed.

[Brief description of the drawings]

FIG. 1 is a line graph of phytase activity versus pH.

FIG. 2 is a line graph of acid phosphatase activity versus pH.

FIG. 3 is a bar graph of the amount of APU produced therein for various transgenic plants.

FIG. 4 is a bar graph of the amount of APU for various transgenic lines.

FIG. 5 is a bar graph of APU amounts for various transgenic lines.

FIG. 6 is a bar graph of the specific activity of APU on various transgenic lines.

FIG. 7 is a bar graph of sugar concentration for various transgenic lines.

FIG. 8 is a bar graph of sugar concentration for various transgenic lines.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 15/09 ZNA C12R 1:91) // (C12N 9/16 (C12N 9/32 C12R 1:91) C12R 1:91) (C12N 9/32 (C12N 9/44 C12R 1:91) C12R 1:91) (C12N 9/44 C12N 5/00 C C12R 1:91) 15/00 ZNAA (71) Applicant 500586738 128, Sec. 2, Academia Road, Nan-Kang, Taipei, 115 Taiwan, R.O. C. (72) Inventor Chen Quo-Joan Bouzyay, Canada 3W 8, Be. C. , Richmond, St. Albums Road 17-7695 (72) Inventor Lu Li-Fai Taiwan Taipei 115, Chin Fast Street, No. 30-4, 7F (72) Inventor Shaw Jay-F Taiwan Taipei 115, Nankan, Jen-Juan Jen Road, Section 2, Array Lane 128, Number 3 F Term (Reference) 2B030 AA02 AB03 AD08 CA06 CA17 CA19 CB02 CD03 CD07 CD09 CD13 CD17 4B024 AA08 BA11 BA12 BA13 CA03 CA04 CA09 DA01 DA05 DA06 EA04 FA02 FA18 GA11 GA14 GA17 GA19 4B050 CC03 DD02 DD13 EE10 LL10 4B065 AA01Y AA88X AA88Y AB01 BA02 BA03 BD01 CA27 CA53

Claims (31)

[Claims] 1. A transgenic plant, wherein the genomic DNA comprises a promoter and a gene encoding a hydrolase and comprising a nucleotide sequence operably linked to the promoter, wherein the promoter is Developing seeds of the transgenic plant,
A transgenic plant that drives expression of the hydrolase in germinating, germinated, or seedling plant seed tissue. 2. The transgenic plant of claim 1, wherein said hydrolase hydrolyzes a substrate naturally present in said plant seed tissue of said transgenic plant. 3. The plant of claim 1, wherein the plant is a rice, barley, rye, oat, or rape plant.
The transgenic plant according to item 1. 4. The transgenic plant according to claim 1, wherein said promoter is an α-amylase gene promoter. 5. The transgenic plant according to claim 4, wherein said promoter is an αAmy8 promoter. 6. The transgenic plant according to claim 1, wherein said promoter is a glutelin gene promoter. 7. The method of claim 1, wherein said enzyme is phytase.
The transgenic plant according to item 1. 8. The transgenic plant according to claim 1, wherein said enzyme is amylopullulanase or α-amylase. 9. A transgenic seed produced from the transgenic plant according to claim 1. 10. The transgenic seed according to claim 9, wherein said seed is germinating. 11. A transgenic seed produced from the transgenic plant according to claim 2. 12. The seed according to claim 11, wherein said seed is germinating.
The transgenic seed according to item 1. 13. A transgenic seed produced from the transgenic plant according to claim 3. 14. The seed according to claim 13, wherein said seed is germinating.
The transgenic seed according to item 1. 15. A transgenic seed produced from the transgenic plant according to claim 4. 16. The seed according to claim 15, wherein said seed is germinating.
The transgenic seed according to item 1. 17. A transgenic seed produced from the transgenic plant according to claim 5. 18. The method according to claim 17, wherein the seed is germinating.
The transgenic seed according to item 1. 19. A transgenic seed produced from the transgenic plant according to claim 6. 20. The seed according to claim 19, wherein the seed is germinating.
The transgenic seed according to item 1. 21. A transgenic seed produced from the transgenic plant according to claim 7. 22. The seed according to claim 21, wherein said seed is germinating.
The transgenic seed according to item 1. 23. A transgenic seed produced from the transgenic plant according to claim 8. 24. The seed according to claim 23, wherein the seed is germinating.
The transgenic seed according to item 1. 25. A method for producing a hydrolase, the method comprising providing a transgenic plant according to claim 1, collecting a transgenic seed from said transgenic plant, and said transgenic plant. Germinating the transgenic seed to produce the hydrolase. 26. The method of claim 25, further comprising isolating said hydrolase from said transgenic seed. 27. A method for producing a polypeptide, comprising the steps of: genomic DNA of a transgenic plant, encoding an α-amylase promoter, and a polypeptide and operably linked to the promoter. Providing the transgenic plant comprising a gene comprising the nucleotide sequence obtained; collecting a transgenic seed from the transgenic plant; germinating the transgenic seed to produce a mature seed; and the mature seed Isolating the polypeptide from the polypeptide. 28. The method of claim 27, wherein in the step of isolating, the polypeptide is isolated from an embryo or endosperm of the mature seed. 29. A method for producing a polypeptide, the method comprising the steps of: transgenic plant genomic DNA encoding a glutelin gene promoter, and a polypeptide, and operably linked to the promoter. Providing the transgenic plant comprising a gene comprising a nucleotide sequence; collecting a transgenic seed from the transgenic plant; germinating the transgenic seed; Isolating the polypeptide. 30. A method for preparing a transgenic plant, comprising the step of transferring to a plant a promoter and a gene encoding a hydrolase and comprising a nucleotide sequence operably linked to the promoter. Wherein the promoter drives expression of the hydrolase in the developing seed, germinating seed, germinated seed or seedling plant tissue of the transgenic plant. Method. 31. A method for preparing a transgenic seed produced from a transgenic plant prepared by the method according to claim 30.